The ability to achieve high-level and long-term recombinant protein expression after gene transfer in skeletal muscle is a crucial issue in the field of gene therapy. Because skeletal muscle is an important tissue that is readily accessible and that is highly vascularized, it could be used as a factory to produce proteins with therapeutic values (reviewed in [1-3]). Indeed, it has been demonstrated that functional therapeutic proteins can be synthesized by the skeletal muscle and secreted into the blood circulation in sufficient amount to mitigate the pathology associated with disorders such as hemophilia, Pompe disease, Fabry's disease, anaemia, emphysema, and familial hypercholesterolemia for example [4-10]. The ability to express recombinant proteins in skeletal muscle is also an important issue for the treatment of neuromuscular disorders such as Duchenne and limb girdle muscular dystrophy. These disorders are caused by mutations of a gene that produces an essential muscle protein. One potential treatment for such disorders is gene transfer, whose objective is to introduce into the muscle a normal and functional copy of the gene that is mutated (reviewed in [11, 12]).
Although high-level of recombinant protein production can be achieved after gene transfer in skeletal muscle using cis-acting DNA elements (promoter and enhancers) derived from viruses [13-16], these regulatory elements are not cell-specific. Besides the safety issues and potential toxicity associated with non-specific protein expression in different tissues of the body, recombinant proteins that are controlled by enhancer/promoter elements derived from viruses are more immunogenic than those produced in a tissue-specific manner, because they are more likely to be expressed in specialized antigen presenting cells [5, 17-20]. As a result, the use of tissue-specific enhancer/promoters, such as those that are active only in skeletal muscle, can prolong and stabilize the production of recombinant protein in vivo. The major drawback of using tissue specific regulatory elements is their strength, which is usually weaker than those derived from viruses [16, 18, 19, 21]. This implies that additional copies of the transgene (or vector carrying it) are necessary to produce sufficient proteins to achieve therapeutic value if a weaker combination of enhancer/promoter is used. In addition to increasing its cost, this may reduce the safety and increase the toxicity of the therapy. An ideal enhancer/promoter combination for gene transfer to skeletal muscle should be strong and muscle-specific. Because the transport capacity of some promising viral vectors is limited, the combination of enhancer/promoter elements carried by these vectors should be relatively small (less than 1-kb). Furthermore, because the skeletal muscle consists of a mixture of fast-twitch and slow-twitch muscle fibers, the enhancer/promoter elements should be active also in both types of fibers to maximize the protein production level.
Troponin I is an abundant component of the thin filament of myofibrils of striated muscle. In adult vertebrate skeletal muscles, two isoforms, the slow (TnISlow) and the fast (TnIFast) isoforms, that are expressed in fast-twitch and slow-twitch muscle fibers and that are encoded by two different genes, exist. The regulatory elements controlling the expression of TnISlow and TnIFast are relatively well characterized. In the case of TnISlow, a small upstream enhancer of about 160-bp (USE) also referred to as SURE, confers slow fiber type specificity [22, 23]. If a fragment of 100-bp is deleted at the 5′ end of USE, the resulting enhancer (ΔUSE) remains muscle specific, but is now able to confer expression in slow-twitch as well as fast-twitch muscle fibers [24]. Specific expression of the TnIFast in fast-twitch muscle fibers is controlled by a 150-bp enhancer known as IRE or FIRE located within the first intron of that gene [23, 25, 26].
Thus, there is a need in the art for strong, muscle-specific regulatory elements for controlling gene transfer and/or expression in muscles.